Peroxisomes are subcellular organelles found in mammalian cells, plant cells and in some eukaryotic microorganisms. Although they are simple organelles, a single membrane surrounds only a few matrix proteins, containing no DNA, their importance is illustrated by the occurrence of human diseases in which there are abnormalities concerning peroxisomes. In Zellweger's cerebroheptorenal syndrome, there is a complete absence of kidney and liver peroxisomes, whereas in cerebrotendinous xanthomatosis there is an increase in the number and size of liver peroxisomes. The long-term objectives of this research are to elucidate the mechanisms involved in peroxisome biogenesis. The large quantities of peroxisomes elaborated when Candida yeast grow on methanol provide a convenient model system to use in this area, because of their ease of isolation, inducibility, and possibility for genetic manipulation. The current proposal focuses on the mechanism of import of cytoplasmically-synthesized alcohol oxidase, a major peroxisomal matrix protein, across the peroxisomal membrane. Specific aims are 1) to purify and characterize an ATPase, recently observed in peroxisomal membranes, that may be crucial to the import process, and 2) to investigate the nature and formation of a complex of newly-synthesized matrix proteins that is formed when import is disrupted by the ionophore carbonylcyanide-m-chlorophenylhydrazone (CCCP). Methodologies planned for the first objective include large-scale isolation of peroxisomes from yeasts, separation of the membranes from these and detergent solubilization of the ATPase. The enzyme will then be purified by ion-exchange, gel-filtration and affinity chromatography methods. The purified enzyme will then be characterized for size, structure, and kinetic properties. For the second objective, the effects of different ionophores on the formation of the complex will be probed by pulse-labelling of spheroplasts with (35S)methionine followed by immunoprecipitation with antiserum against AO and dihydroxy acetone synthetase, the other major matrix protein. The composition and size of the complex will be determined by density centrifugation and SDS-gel electrophoresis. Future research will use chemeric proteins constructed using recombinant DNA methodology to probe structural features of matrix proteins important to assembly.